Heterodimeric cytokine receptors comprise two (or three) subunits which subserve distinct and specialised functions. These include a major ligand-binding subunit (the α subunit) and a signalling subunit (the β or γ subunit). Importantly, the latter is able to recognise several cytokines complexed to the appropriate α chain and transduce their signals. This is exemplified by the common β chain (βc) of the human granulocyte-macrophage colony-stimulating factor GM-CSF, interleukin-3 (IL-3) and IL-5 receptors, the common IL-2 receptor γ chain (shared by the IL-2, IL-4, IL-7, IL-9 and IL-15 receptors) and gp130 (shared by the IL-6, IL-11, LIF, ciliary neutrophic factor, oncostatin M and cardiotrophin receptors). Significantly, IL-5, IL-3 and GM-CSF, the only three cytokines known to stimulate eosinophil production, can be found concomitantly elevated in lungs affected by allergic inflammation.
The simultaneous antagonism of all three GM-CSF, IL-3 and IL-5 may be desirable or indeed necessary for stimulating eosinophils. For example, eosinophils which are believed to be the major cell type involved in allergy can be maintained in numbers and be stimulated by either IL-3, GM-CSF or IL-5 (Lopez et al, 1989). Antagonism of all three cytokines may thus be necessary to inhibit the actions of eosinophils and basophils. Similarly, basophils which are also believed to play an effector role in allergy can be stimulated by either IL-3, GM-CSF or IL-5 (Lopez et al, 1990). Antagonism of GM-CSF, IL-3 and IL-5 may be accomplished by the concomitant administration of specific antagonists for each different cytokine. Though feasible, this approach has the disadvantage of having to administer up to three different proteins which is not only inconvenient but which also increases the risk of immunogenicity and other side-effects.
Because all three of these cytokines act through a common receptor subunit (βc) it may be possible to simultaneously inhibit the action of GM-CSF, IL-3 and IL-5 with a single compound via the (βc) subunit.
Thus, an antagonist directed against the βc chain may simultaneously inhibit the function of all three cytokines and may prove a useful therapeutic.
One of the major problems in seeking structural data of the binding site of a communal subunit complexed to cytokines is that, unlike homodimeric receptors or isolated α chains of heterodimeric receptors which can directly bind to cytokines, communal subunits cannot bind to cytokines by themselves. To overcome this problem applicants have developed a monoclonal antibody (Mab) against a region which is important for cytokine high affinity binding within domain 4 of the GM-CSF/IL-3/IL-5 common beta chain (D4βc) receptor. This Mab, termed BION-1, inhibited the high affinity binding of GM-CSF, IL-3 and IL-5 to human eosinophils, and inhibited their in vitro production and functional activation. BION-1 thus represents the first common antagonist of the GM-CSF, IL-3 and IL-5 receptors and a unique tool with which to explore the cytokine-binding site in the common beta chain.
The molecular basis for the affinity conversion of βc to each ligand is not fully understood as the ligand-receptor complex had not yet been crystallised and this has prevented the structural definition of their ligand-binding sites. Applicants have now crystallised and determined the structure of the D4βc domain of the GM-CSF/IL-3/IL-5 receptor bound to an antagonist in the form of BION-1.